Otitis media (OM) is the second most common disease of childhood, and an important OM pathogen that can cause both acute and recurrent disease is nontypeable Haemophilus influenzae (NTHi). We have identified a toxin-antitoxin protein pair in NTHi, VapBC-1, as being crucial for the ability of the organism to survive during experimental OM. Upon encountering stressful conditions, the VapB-1 antitoxin is degraded and the VapC-1 ribonuclease toxin is freed to cleave mRNA, arresting bacterial growth. This state of dormancy facilitates nonspecific antibiotic tolerance, as most antimicrobials are designed to inhibit essential cellular functions necessary for replication. Further, it allows a subpopulation f the infecting bacteria to survive treatment and resume growth, resulting in recurrent disease. Homologues of this protein pair are very highly conserved in numerous bacterial genomes. We intend to target this protease-mediated molecular switch and block the ability of the organism to go into stasis using a high-throughput screening approach to discover and validate novel chemical probes that interfere with VapC-1 toxin activity. These small molecule probes will inhibit the ability of the organism to enter into a bacteriostatic state, allowing antimicrobial therapy to kill the bacteria that were previously tolerant due to dormancy. We propose the following Specific Aims: 1) Use our innovative miniaturized and validated fluorescence-quenching ribonuclease interference assay to screen the NCATS library of small molecules for toxin-targeted compounds (TTCs); 2) perform secondary screens against the hits including a different detection wavelength, a specificity assay and a cytotoxicity assay; 3) validate the specificity of the non-cytotoxic TTCs in vitro using an engineered bacterial strain with inducible VapC-1 toxin expression, and 4) determine the effectiveness of the TTCs to treat NTHi infections of primary human respiratory tissues held at the air-liquid interface. Treatment with the chemical probes validated in this study could be used in conjunction with antibiotics to achieve a complete eradication of infecting organisms, giving clinicians a novel adjunctive therapy for their patients with recurrent disease.